Both the retinoblastoma gene (RB) and transcription factor E2F play a critical role in cell growth control (for a review, see Adams, P. and Kaelin, W. Seminars in Cancer Biology 6:99-108 (1995)). The RB locus is frequently inactivated in a variety of human tumor cells. Reintroduction of a wild-type RB gene (e.g., Bookstein et al. Science 247:712-715 (1990)) or RB protein (pRB) (e.g., Antelman et al. Oncogene 10:697-704(1995)) into RBneg/RBmut cells can suppress growth in culture and tumorigenicity in vivo.
While E2F serves to activate transcription of S-phase genes, its activity is kept in check by RB. RB arrests cells by blocking exit from G into S-phase (for example, Dowdy et al. Cell 73:499-511 (1993)) but the precise pathway of the arrest remains unclear.
Although E2F forms complexes with RB, complex formation is more efficient if an E2F-related protein, DP-1, is present. E2F-1 and DP-1 form stable heterodimers which bind to DNA (for example, Qin et al. Genes and Dev. 6-:953-964 (1992)). DP-1-E2F complexes serve to cooperatively activate transcription of E2F-dependent genes. Such transcription can be repressed by pRB in the same manner as E2F-1 or DP-1 activated transcription.
Transcriptional repression of genes by RB in some instances can be achieved by tethering pRB to a promoter. For example, GAL4-pRB fusions bind to GAL4 DNA binding domains and repress transcription from p53, Sp-1 or AP-1 elements (Adnane, et al. J. Biol. Chem. 270:8837-8843 (1995); Weintraub, et al. Nature 358:259-261 (1995)). Sellers, et al. (Proc. Natl. Acad. Sci. 92:11544-11548 (1995)) disclosed fusions of amino acid residues 1-368 of E2F with amino acids 379-792 or 379-928 of RB.
Chang, et al. (Science 267:518-521 (1995)) disclosed the use of a replication-defective adenovirus-RB construct in the reduction of neointima formation in two animal models of restenosis, a hyperproliferative disorders.
The instant invention provides the surprising result that a-fusion of an E2F polypeptide with an RB polypeptide is more efficient in repressing transcription of the E2F promoter than RB alone, and that such fusions can cause cell cycle arrest in a variety of cell types. Such fusions can thus address the urgent need for therapy of hyperproliferative disorders, including cancer.
One aspect of the invention is a polypeptide comprising a fusion of a transcription factor, the transcription factor comprising a DNA binding domain, and a retinoblastoma (RB) polypeptide, the RB polypeptide comprising a growth suppression domain. Another aspect of the invention is DNA encoding such a fusion polypeptide. The DNA can be inserted in an adenovirus vector.
In some embodiments of the invention, the transcription factor is E2F. The cyclin A binding domain of the E2F can be deleted or nonfunctional. The E2F can comprise amino acid residues about 95 to about 194 or about 95 to about 286 in some embodiments.
The retinoblastoma polypeptide can be wild-type RB, RB56, or a variant or fragment thereof. In some embodiments, the retinoblastoma polypeptide comprises amino acid residues of about 379 to about 928. Preferred amino acid substitutions of the RB polypeptide include residues 2, 608, 788, 807, and 811.
Another aspect of the invention is an expression vector comprising DNA encoding a polypeptide, the polypeptide comprising a fusion of a transcription factor, the transcription factor comprising a DNA binding domain, and a retinoblastoma (RB) polypeptide, the RB polypeptide comprising a growth suppression domain. In some embodiments a tissue-specific promoter is operatively linked to DNA encoding the fusion polypeptide. The tissue-specific promoter can be a smooth muscle alpha actin promoter.
Another aspect of the invention is a method for treatment of hyperproliferative disorders comprising administering to a patient a therapeutically effective dose of an E2F-RB fusion polypeptide. The hyperproliferative disorder can be cancer. In some embodiments the hyperproliferative disorder is restenosis. The fusion polypeptide and nucleic acid encoding the fusion polypeptide can be used to coat devices used for angioplasty.